is subjected to freeze-thaw stress in commercial processes, including frozen dough

is subjected to freeze-thaw stress in commercial processes, including frozen dough baking. tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. Yeast (wild-type strain BY4741 (deletion strains derived from BY4741 were obtained from EUROSCARF (the European Archive for Functional Analysis). YPD medium (1% yeast extract [Difco Laboratory, Detroit, MI], Tarafenacin 2% peptone [Difco Laboratory], and 2% glucose) was utilized for cultivation of the yeast cells unless normally noted. SD medium without copper (2% glucose, 0.17% yeast nitrogen base without Cu and Fe [Bio 101, Vista, CA], 0.5% ammonium sulfate, amino acid, and 1 M ferric chloride) supplemented with various concentrations of copper ions (0 M, 1 M, or 10 M CuSO4 alone Tarafenacin or 10 M CuSO4 plus 100 M copper chelator bathocuproinedisulfonic acid disodium salt [BCS]) was utilized for measurement of the intracellular ROS level and SOD activity. Viable numbers of yeast cells were decided using YPD medium solidified with 2% agar. Cell suspensions were plated onto the agar plates with appropriate dilution and produced for 2 days at 30C to allow colony formation, and viable cell numbers were expressed as CFU. Viability was represented by the ratio of viable cell figures after freeze-thaw treatment to viable cell figures before freeze-thaw treatment. The viability of cells before freeze-thaw treatment was taken as 100%. Freeze-thaw treatment condition. The candida cells were cultivated at 30C with shaking at 200 rpm until the log growth phase (optical denseness at 600 nm [OD600] of 1 1) in YPD medium. The cells were harvested by centrifugation, washed in distilled water, and resuspended in an equal volume of distilled water at room heat. An aliquot of 5 ml was then taken from the cell suspensions and used as a negative control (nonfrozen control). The remaining cell suspensions were divided into 5-ml aliquots CACNB4 and frozen at ?30C for 24 h, 48 Tarafenacin h, 72 h, or 96 h. The frozen cell suspensions were then thawed at 30C inside a water bath for 10 min. Measurement of intracellular levels of ROS. The intracellular levels of ROS were measured using the oxidant-sensitive probe 2,7-dichlorofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR). After freeze-thaw treatment, the candida cells were collected and resuspended in YPD medium, and then the cell tradition was incubated at 30C for 60 min (postthaw incubation). Thirty minutes before the end of postthaw incubation, DCFDA (final concentration at 10 M) was added to the cell tradition. The cells were then washed, resuspended in 200 l of distilled water, and disrupted with glass beads inside a FastPrep FP100A instrument (Bio 101) for 40 s in the rate establishing of 6.5. Cell components (50 l) were mixed with 450 l of distilled water, and the fluorescence was measured at emission = 538 nm (excitation = 485 nm) using a SpectraMax Tarafenacin Gemini XS microplate spectrofluorometer (Molecular Products, Sunnyvale, CA). The ideals of emission = 538 nm were normalized using the viable cell figures. The Tarafenacin fluorescence intensity of the nonfrozen control cells was relatively taken as 100%. DNA microarray analysis of indirect gene manifestation. We performed DNA microarray analysis of indirect gene manifestation as follows. The candida cells were exposed to freeze-thaw treatment for 0, 24, or 48 h, and then the cells were subjected to postthaw incubation for 60 min at.